Most hormones and neurotransmitters bind to cell surface receptors, activate G proteins, and produce a second messenger that propagates the signal within the cell. In yeast the G-beta/gamma subunits are necessary and sufficient for most aspects of the response to mating pheromones. Recently however,we determined that the G-alpha protein (Gpa1) can also transmit a signal mediated by the only known phosphatidylinositol 3-kinase in yeast (Vps34) and the phospholipid second messenger, Ptdlns 3-P. Notably, Gpa1 and Vps34 are both located at the endosome, far removed from receptors and the G-beta/gamma subunits. Our objective in this proposal is to identify other components of the pathway (receptor, G- beta/gamma, effector target) and the mechanism by which these proteins transmit their signal at the endosome. There are three specific aims: Aim 1. How is the G protein at the endosome activated? We have shown that Gpa1 stimulates Vps34 and Ptdlns 3-P production at the endosome but we do not know how Gpa1 is activated in this case. Our hypothesis is that Gpa1 and Vps34 are activated by internalized pheromone receptors and subsequently by a second exchange factor at the endosome. Aim 2. What are the G-beta/gamma-like proteins at the endosome? We found that the Ptdlns 3-kinase regulatory subunit Vps15 has a 7-WD repeat domain structure typical of that found in G-beta proteins. Our hypothesis is that Vps15 functions as the G-beta subunit at the endosome. Aim 3. How is G protein signaling at the endosome transmitted? We have shown that the Ptdlns 3-P- binding protein Bem1 translocates to the endosome in response to Gpa1 activation. Our hypothesis is that Ptdlns 3-P activates Bem1 and recruits a subset of signaling proteins to the endosome. These findings reveal a surprising new role and a new site of action for this well-characterized signaling molecule. Mechanisms discovered in yeast are usually recapitulated in higher eukaryotes, so the principles elucidated here will likely apply as well to hormone and neurotransmitter function in humans. Just as G protein coupled receptors at the cell surface are proven targets for the treatment of hypertension and diverse neurological disorders, intracellular receptors are likely as well to be useful in treating human disease.